The objective of this application is to elucidate the functions of 1a, 25-dihydroxyvitamin D3 (1a,25(OH)2D3) and its receptor, the 1a,25(OH)2D3 receptor (VDR), in tissues in which it does not have a role in trans-epithelial calcium (Ca) and phosphate (Pi) transport ("non-classical" effects) using the zebrafish, Danio rerio as a model organism. We have recently shown that the VDR is expressed in tissues of the adult and developing zebrafish, Danio rerio. The VDR is present in the developing cartilage, bone, brain, eye, and ear of the zebrafish early after fertilization. The hypothesis is that 1a,25(OH)2D3 and the VDR alter cartilage development and maturation in zebrafish by regulating the expression of bone morphogenetic proteins (BMPs) and BMP receptors. In aim 1 we will examine the effects of 1a,25(OH)2D3 on skeletal development in Danio rerio. We will administer 1a,25(OH)2D3 to developing zebrafish embryos to determine whether 1a,25(OH)2D3 administration alters BMP and BMP receptor expression, BMP signaling and cartilage development and skeletal morphology. In aim 2, we will determine the effects of "knocking down" VDR expression in the developing zebrafish embryo with anti-sense morpholino oligonucleotides methods. The effect of VDR knock- down on zebrafish embryo BMP and BMP receptor expression, BMP signaling and cartilage development and skeletal morphology will be assessed. Significance: The effects of 1a,25(OH)2D3 and its receptor have been extensively examined in mammals and humans with respect to their effects on Ca transport. Effects that are independent of Ca transport have not, however, been examined in detail. By examining the effects of 1a,25(OH)2D3 and the VDR on skeletal development in zebrafish embryos through BMP pathways, we will be able to delineate the direct and Ca-independent effects of 1a,25(OH)2D3 and the VDR in cartilage. We will also establish the zebrafish as a new model organism in which to examine the effects of 1a,25(OH)2D3. PUBLIC HEALTH RELEVANCE: Vitamin D is important for the maintenance of bone health. In this project we will examine how the vitamin and its receptor function in bone and cartilage by studying their effects in a model organism, the developing zebrafish. By examining the effects of active vitamin D and its receptor on growth factors that influence skeletal development we will gain insights into how the vitamin functions in bone. This information will be of use in preventing bone loss that occurs with aging and in treating osteoporosis.